Proteoglycans are major structural components of the extracellular matrix of cartilage where they are found largely organized into aggregates. These aggregates consist of a number of proteoglycan monomers noncovalently bound to hyaluronic acid in an interaction stabilized by a link protein. These molecules are responsible for the resiliency of the tissue, a property required for normal function. Recent studies on the biosynthesis of proteoglycans and their subsequent assembly into aggregates in a model system of primary cultures of chondrocytes from a transplantable rat tumor have yielded much information about proteoglycan synthesis but much less about link protein. This proposal is directed at extending the previous studies in the rat chondrosarcoma model system with emphasis on learning more about the biosynthesis of link protein. Labeling experiments will be done to determine the relative rates of link protein and proteoglycan core protein synthesis. The radioactivity in the newly synthesized link and core proteins will be determined by immunoprecipitation with rabbit antiserum to link protein or core protein or by reaggregation with carrier proteoglycan and subsequent purification. The intracellular half-time for newly labeled link protein relative to core protein will be determined. Currently, immnological methods will be developed to allow the quantitation of link protein in the presence of the other components of the aggregate. These methods then will be employed in the isolation and characterization of a monomer-link protein complex and conditions will be determined which will prevent the formation of such a complex but will not cause its disassembly. These conditions in combination with the appropriate labeling times previously determined will then be used to examine the formation of monomer (or precursor core protein)-link protein complexes and to determine whether such complex form intracellularly. These studies will extend our knowledge of the assembly process for the formation of proteoglycan aggregates and may offer clues into the mechanisms by which cells may control proteoglycan aggregation, a self assembly process occurring some distance away from the site of synthesis in chondrocytes.